Cytometry Part B (Clinical Cytometry) 90B:31–39 (2016)

Original Article
Consensus Guidelines on Plasma Cell Myeloma
Minimal Residual Disease Analysis and Reporting
Maria Arroz,1* Neil Came,2 Pei Lin,3 Weina Chen,4 Constance Yuan,5 Anand Lagoo,6
Mariela Monreal,7 Ruth de Tute,8 Jo-Anne Vergilio,9 Andy C. Rawstron,10
and Bruno Paiva11
1
Department of Clinical Pathology, Cytometry Laboratory, CHLO, Hospital S. Francisco Xavier, Lisbon, Portugal
2
Pathology Department, Peter MacCallum Cancer Centre, Melbourne, Australia
3
Department of Hematopathology, MD Anderson Cancer Center, Houston, Texas, USA
4
Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas, USA
5
Laboratory of Pathology, NCI, NIH, Bethesda, Maryland, USA
6
Department of Pathology, Duke University Medical Center, Durham, North Carolina, USA
7
Interpflow Corporation, Miami, Florida, USA
8
HMDS, Department of Haematology, St. James’s Institute of Oncology, Leeds, United Kingdom
9
University Of Michigan Medical Center Hematology Oncology Laboratory, Ann Arbor, Michigan, USA
10
HMDS, Department of Haematology, St. James’s Institute of Oncology, Leeds, United Kingdom
11
Clinica Universidad de Navarra, Centro de Investigaci
on Medica Aplicada, University of Navarra, Pamplona, Spain

Background: Major heterogeneity between laboratories in flow cytometry (FC) minimal residual disease
(MRD) testing in multiple myeloma (MM) must be overcome. Cytometry societies such as the International Clin-
ical Cytometry Society and the European Society for Clinical Cell Analysis recognize a strong need to establish
minimally acceptable requirements and recommendations to perform such complex testing.
Methods: A group of 11 flow cytometrists currently performing FC testing in MM using different instru-
mentation, panel designs ( 6-color) and analysis software compared the procedures between their
respective laboratories and reviewed the literature to propose a consensus guideline on flow-MRD analy-
sis and reporting in MM.
Results/Conclusion: Consensus guidelines support i) the use of minimum of five initial gating parameters
(CD38, CD138, CD45, forward, and sideward light scatter) within the same aliquot for accurate identification of
the total plasma cell compartment; ii) the analysis of potentially aberrant phenotypic markers and to report the
antigen expression pattern on neoplastic plasma cells as being reduced, normal or increased, when compared
to a normal reference plasma cell immunophenotype (obtained using the same instrument and parameters);
and iii) the percentage of total bone marrow plasma cells plus the percentages of both normal and neoplastic
plasma cells within the total bone marrow plasma cell compartment, and over total bone marrow cells. Consen-
sus guidelines on minimal current and future MRD analyses should target a lower limit of detection of
0.001%, and ideally a limit of quantification of 0.001%, which requires at least 3 3 106 and 5 3 106 bone
marrow cells to be measured, respectively. VC 2015 International Clinical Cytometry Society

Key terms: plasma cell myeloma; minimal residual disease; flow cytometry

How to cite this article: Arroz M, Came N, Lin P, Chen W, Yuan C, Lagoo A, Monreal M, de Tute R, Vergilio
J.-A, Rawstron A C. and Paiva B. Consensus Guidelines on Plasma Cell Myeloma Minimal Residual Disease Analy-
sis and Reporting. Cytometry Part B 2016; 90B: 31–39.

*Correspondence to: Maria Arroz, Department of Clinical Pathology, Received 9 December 2014; Revised 15 January 2015; Accepted
Cytometry Laboratory, CHLO, Hospital S. Francisco Xavier, Estrada do 16 January 2015
Forte do Alto do Duque, 1495-005 Lisbon, Portugal. E-mail: marroz@ Published online 2 June 2015 in Wiley Online Library
chlo.min-saude.pt (wileyonlinelibrary.com).
DOI: 10.1002/cyto.b.21228

C 2015 International Clinical Cytometry Society

V
32
FIG. 1. Initial gating strategy for identifying the BM PC compartment. A relevant parameter is plotted against time as a measure of the quality and con-
tinuity of data acquired for the whole sample (A). Light scatter characteristics are used to exclude doublets (B and C) and debris (D). All possible plasma
cells are captured by generous gating on CD381 and CD1381 bright events (E) and CD381 bright events plotted against CD45 (F).
The principles outlined in the Practice Guidelines that
were recently published by the International Council for
Standardization in Haematology (ICSH) and International
Clinical Cytometry Society (ICCS) (1) guided an interna-
tional working group of experts in the field, under the
auspices of the ICCS and the European Society for Clini-
cal Cell Analysis (ESCCA) toward consensus on: analysis
and gating strategy, data interpretation, reporting con-
ventions, and a minimum set of recommendations that
can be applied in the optimal assessment of minimal
residual disease (MRD) in multiple myeloma (MM) using
multidimensional flow cytometry (FC).
An increasingly higher number of flow cytometry lab-
oratories use digital instruments that can accommodate
eight or more colors. These provide superior event
acquisition and analysis of a greater number of cells uti-
lizing more parameters per tube than was previously fea-
sible with four color instruments. Six well-chosen colors
in a single tube represent the minimum number recom-
mended for low-sensitive flow-MRD analysis in MM. Simi-
larly, all currently available analysis software packages
enable the drawing of regions (“gates”) allowing the par-
titioning of data into cell subsets in a relatively user-
friendly, stepwise, accurate, and reproducible way. More
sophisticated “downstream” algorithms that require
rapid execution of complex mathematical transforma-
ARROZ ET AL.
tions such as sequential or Boolean gating, hierarchical
clustering, or principle component analysis (PCA) are
now commonly available, and will further help on iden-
tifying rare plasma cell (PC) events, in a reproducible
way, within pluricellular bone marrow (BM) samples.
FLOW CYTOMETRY DATA ANALYSIS AND PLASMA CELL
GATING STRATEGY
In adopting a “fit for purpose” gating strategy for iden-
tifying potentially rare PC events it is important to use a
gating strategy which minimizes subjectivity and maxi-
mizes reproducibility (1). Inicial gates to identify PCs
and discriminate them from all other BM cells are drawn
generously enough to include as many PCs events as
possible, allowing for variants that express lower CD38,
CD45 and/or CD138 to be included, while excluding
contaminating lymphocytes, doublets and other cellular
debris (Fig. 1).
Overall despite different instruments and variable
flow-MRD panel designs (6, 8, or 10-color) between lab-
oratories, the adoption of the European Myeloma Net
(EMN) approach using a minimum of five gating parame-
ters (CD38, CD138, CD45, FSC, and SSC properties)
within the same tube for the identification of the total
PC compartment is recommended (2). This maximizes
the inclusion of normal and neoplastic PCs whilst, at
Cytometry Part B: Clinical Cytometry
 GUIDELINES ON MYELOMA MRD ANALYSIS AND REPORTING 33

FIG. 2. Expression of routinely used antigens on bone marrow aspirates. A: Erythroblasts and leukocytes by SSC vs. CD45, (B) Normal B-cells/hem-
atogones by SSC vs. CD19; (C) CD27 expression on memory T and B-cells by SSC vs. CD27; (D) CD81 expression on B-cells/hematogones by SSC
vs. CD81; (E) NK cells by SSC vs. CD56; (F) mast cells, myeloid, and early erythroid progenitors by SSC vs. CD117.

the same time, allowing removal of contaminating non- exclusion since both normal and neoplastic PCs can
PC events. (2). show variable CD45 expression.

Initial Gating Strategy

The recommended gating steps are as follows (Fig. 1):
i. Use of a key parameter (e.g., CD38) versus time to
assess the quality and continuity of data acquired,
which provides, for example, the opportunity to gate
out air bubbles and clogs, thus eliminating periods of
time of unstable flow (A).
ii. Use of light scatter to exclude doublets, (FSC-Height
vs. FSC-Area) (B), (SSC-Height vs. SSC-Area) (C), and
debris, (SSC-Area vs. FSC-Area) (D) while exercising
caution not to exclude, from initial gating, hyperdi-
ploid or tetraploid PCs which exhibit aberrantly high
FSC-Area and SSC-Area, but, e.g. low, FSC-Height.
iii. Generous gating is then performed to encompass all
potential CD381 and CD1381 PCs (E), which is fur-
ther refined by incorporating all CD381 bright
events plotted against CD45 (F). CD45 is helpful for
gating but should not be used as a marker for cell
Further Characterization of PCs
Albeit heterogeneous, the normal BM PC immunophe-
notype is highly conserved in normal bone marrow sam-
ples, as well as between individual patient samples at
different disease stages, except when antibody-based
therapies have been used (e.g.: anti-CD38; 3). In this
regard, based on merged data files from different sam-
ples, followed by principle component analysis of the
immunophenotype of normal BM PCs (as assessed by up
to 12 different markers combined in two eight-color
tubes (CD45, CD138, CD38, CD19, CD56, or CD28,
B2M, or CD27, CyIgj or CD117, CyIgk or CD81), the
Euroflow Consortium demonstrated that BM PCs from
healthy donors, patients with non-PC related diseases, or
regenerating BM following chemotherapy in patients
with non-PC related diseases, had fully overlapping and
highly similar immunophenotypes (3). In addition, such
normal PC immunophenotypes did not overlap with

Cytometry Part B: Clinical Cytometry

34
FIG. 3. Normal and neoplastic PCs coexisting within the BM PC compartment. A and B demonstrate the combined analysis gate strategy to identify
all PCs. According to multiple abnormalities in antigen expression, neoplastic PCs (black) and normal PCs (grey) can be identified in subsequent dot
plots for CD56 vs. CD38 (C), CD45 vs. CD19 (D), CD81 vs. CD117 (E), and CD38 vs. CD27 (F).
that of >97% of neoplastic PCs from a large series of
MM patients studied at diagnosis.
That notwithstanding, it should be noted that appropri-
ate multivariate analysis with  8 markers is required to
reach such an applicability because it is now well
recognized that minor subsets of normal BM PCs can
express some markers with intensity levels previously
considered to be aberrant (4,5,6). In this regard, Liu et al.
(5) and Tembhare et al. (6) have proposed some helpful
insights that can assist in the discrimination between nor-
mal versus neoplastic PCs, as described below.
Normal PCs show heterogeneous expression of CD19
and CD45, they are usually negative for CD20 and CD117,
and they invariably show homogeneously bright expres-
sion of CD81 (6). In turn, CD28 and CD56 expression is
present in a small subset (between 5–20%) of normal PCs.
Accordingly, normal PCs typically show heterogeneous or
bimodal expression for some of the most commonly used
markers (e.g. for CD19, CD56, and CD45) while neoplas-
tic PCs from MM patients usually have more homogene-
ous antigen expression. Neoplastic PCs will often show
additional aberrancies, such as CD117 expression, weaker
CD38 expression, and increased FSC and/or SSC features.
Noteworthy, such aberrancies will be co-expressed within
the same cell population, whereas immunophenotypic
variations in normal PCs tend to be heterogeneous and/or
distributed amongst different well-defined subsets. Pro-
vided a sufficient number of PCs are acquired, analysis of
cytoplasmic light chain expression within specific PCs
ARROZ ET AL.
subsets showing myeloma-like aberrant phenotypes (e.g.
CD19-negative or CD56-positive PCs), can be a valuable
second step to confirm the normal vs. aberrant nature of
such PCs in a minor subset of cases.
Even though antigenic shift upon therapy cannot be
precluded (7), major antigenic shifts with respect to the
consensus markers were typically absent in large
cohorts in the Medical Research Council Myeloma IX
Study (8). However, it is important to note that the base-
line tumor clone often shows also phenotypic heteroge-
neity, and that all different phenotypic subclones should
be followed throughout therapy (9). In fact, it has been
recently shown that the distribution of such subclones
may fluctuate from diagnostic into MRD samples, but
this should be interpreted as clonal selection rather than
antigenic shift upon therapy (9).
Defining Neoplastic PCs
No single parameter reliably distinguishes neoplastic
PCs from normal PCs; however, even a limited (six
color) combination of antibodies in a single tube is able
to achieve this in a high proportion of cases provided a
sufficient number of cellular events are evaluated in the
flow cytometer (8). The comparison between the
patient’s sample vs. a normal PC immunophenotype ref-
erence obtained using the same instrument and parame-
ters may be highly valuable. Similarly, internal positive
(e.g., B-cells for CD19 and CD81, NK cells for CD56,
myeloid progenitors and mast cells for CD117) and
Cytometry Part B: Clinical Cytometry
 GUIDELINES ON MYELOMA MRD ANALYSIS AND REPORTING 35

FIG. 4. A: Example of a principle component analysis (PCA) guided approach as the starting point for the separation of a homogeneous PC popula-
tion (black) from the remaining bone marrow cells (grey) traced onto CD38 vs. CD138 (B) and CD38 vs. CD45 (C) dot plots, using the same sample
as depicted in Figure 1. D: Another example of PCA, in this case separating neoplastic PCs (black) from normal PCs (grey) restricted to the total pre-
viously gated bone marrow plasma cell compartment, as demonstrated in figure 3 A and B, traced onto a bivariate plot (E) analyzing CD38 vs.
CD27.

negative control (e.g., erythroblasts for CD45) cell popu-
lations represent a highly reliable strategy to define nor-
mal vs. aberrant phenotypic profiles. This should also be
performed using the patient’s sample for each relevant
antigen as illustrated in Figure 2.
The 2006 Bethesda International Consensus Recommen-
dations advise on the reporting of antigen expression as
being reduced, normal, or increased in intensity compared
with that expressed on appropriate reference cell popula-
tions (10). Reporting in this manner provides a clearer pic-
ture of the immunophenotypic deviation from normal PC
immunophenotype and minimizes the impact of variability
in reagent and instrument performance between laborato-
ries. Reporting should include the percentage of positive
cells for each marker within the specific neoplastic PC
compartment.
The availability of patient-specific diagnostic immuno-
phenotypes may also represent a valuable aid while per-
forming flow-MRD monitoring. The use of polychromatic
(8-color) flow cytometry strategies facilitates this
approach (11).
Perhaps even more useful, particularly in the setting of
multicenter trials, is the availability of a reference library
of normal and neoplastic BM PCs that have been immuno-
phenotyped using the exact same panel of monoclonal
antibodies, under identical sample preparation proce-
dures and conditions (12). In order to accommodate the
degree of intra and inter-patient phenotypic heterogeneity
of normal PCs, the larger the library of cases, the better. A
minimum number of twenty reference cases (for which
similar cell numbers to those of the questioned samples
have been evaluated), is recommended. The discrimina-
tion between normal PCs and neoplastic PCs on pheno-
typic grounds has to be multiparametric, i.e. the identifi-
cation of a neoplastic PC population should never be
based on the isolated deviation of a single marker.
As described above, analysis of the CyIgj/CyIgk
plasma cell ratio within specific PC subsets defined on
the basis of other surface markers (e.g.: CD19- CD45-
CD561) may be informative to confirm the clonal vs.
polyclonal nature of such PC subsets in selected cases.
Summary of the Most Frequent Normal and Neoplastic PC
Immunophenotypes, and of Less Frequent Phenotypes
Observed on Minor Subsets of Normal Plasma Cells
The most common immunophenotype of normal PCs
could be described as CD381bright, CD1381bright,
CD191, CD451, CD202, CD271, CD282, CD562,
CD811, CD1172, CD2002/1, with a polyclonal CyIgj/
CyIgk ratio. Minor subsets of normal PCs (typically

Cytometry Part B: Clinical Cytometry

36 ARROZ ET AL.
representing <30% of total normal PCs) exhibit less
“typical” antigen expression patterns such as: CD192,
CD452/low, CD201, CD27low, CD281, CD561 and
CD2001 bright. (2,13,14).
In contrast, neoplastic PCs show aberrant phenotypes
consisting of multiple possible combinations of the follow-
ing aberrant antigenic profiles: CD38 low (dimmer than
normal PCs), CD192, CD452, CD201, CD272/low,
CD281, CD561/bright, CD812/low, CD1171, CD200
bright, in association with monoclonal CyIgj or CyIgk.
Figure 3 illustrates an example where coexisting normal
and neoplastic PCs within the same bone marrow, display
distinct phenotypic features.
The role of new/more advanced computational tools to
facilitate data analysis for flow-MRD in MM
Traditional visualization of flow cytometric data in
bivariate (2D) dot plots results in an increasingly large
number of scattergrams and a “geometric increase in the
informational content” as the number of antibodies used
increases (10). This is a particular challenge for users
becoming familiar with 8 color flow cytometry and
requires the judicious use and graphical representation of
all potentially relevant 2D combinations of antigen
expression patterns. There is a risk that by relying on vis-
ual interpretation of two-dimensional plots alone the
operator may not fully appreciate all relevant antigen
expression patterns within a particular cell population,
nor gain full insight into the variation of normal antigen
expression profiles. In recent years, a variety of new
computational tools have been developed to identify spe-
cific cell populations in multidimensional flow cytometry
data sets (15,16). One such solution is the use of princi-
pal component analysis (PCA) of a unified data file with a
potentially unlimited number of dimensions (17). A PCA-
based approach is implemented in Infinicyt software in
the automatic population separator (APS) view, where
the first (x axis) and second (y axis) principal compo-
nents are used to produce a bidimensional representation
of either cell clusters or patient profiles; each principal
component is represented by a linear combination of all
parameters included in the FCS file(s) under analysis, as
examplified in Figure 4. The power of this interpretation
method is that it facilitates the development of a normal
reference library of chosen cases, automated separation
of populations within a sample taking into consideration
all parameters, and prospective detection and tracking of
any aberrant population that deviates from the normal
cells. This can be particularly useful in cases with differ-
ent subclones that can be difficult to identify when only
bi-dimensional dot plots are used (9).
While not all users have practical experience with
these new multidimensional data analysis tools and
approaches, or they are not presently resourced to
adopt it, automated multivariate (e.g. PCA-guided) data
analysis approaches for fast identification and characteri-
zation of homogeneous cell populations, coupled with
reference (normal and tumor) databases, in the context
of full technical standardization, may provide automated
classification of normal vs. malignant PCs, and be an
ideal adjunct towards myeloma MRD assessment and
standardization by flow cytometry.
REPORTING CONVENTIONS
Reporting the overall immunophenotypic signature of
neoplastic PCs
Reporting an antigen expression pattern on neoplastic
PCs on an individual marker basis as being reduced, nor-
mal or increased, compared to a normal (reference) PC
immunophenotype (obtained using the same antibody-
reagent combinations, instruments and sample prepara-
tion protocols and conditions), including the percentage
of positive cells for each marker, is recommended to
establish the immunophenotypic signature that will aid
in follow-up MRD detection.
Reporting a sample as MRD positive, MRD negative, or
unsuitable for MRD assessment
The guidelines of the EMN in 2008 proposed that an
MRD negative BM is defined as no detectable abnormal
PCs at a level of sensitivity of at least 0.01%. However
this is on the proviso that the BM aspirate sample is
deemed suitable using the criteria defined earlier, by vis-
ualizing all BM cells simultaneously (Fig. 2). An MRD-
negative bone marrow, in the appropriate clinical con-
text, has also been termed as immunophenotypic
response (IR) or flow-Complete Response (F-CR) (18).
However, the threshold used to distinguish MRD-positive
from MRD-negative may vary from sample to sample,
and is subject to change as technology and treatments
improve; therefore, it is recommended to report on the
level of MRD and the assay limit of detection in addition
to classifying patients as MRD-negative vs. MRD-positive.
At present, several clinical studies have validated the
prognostic value of myeloma flow-MRD monitoring. In
some studies, the presence of MRD was evaluated quanti-
tatively as percent of neoplastic PCs from all BM
nucleated cells (e.g., CD451 leucocytes plus CD45- cellu-
lar BM events such as nucleated red bood cells) (19,20).
Other studies have used the number of total leukocytes,
defined as PCs plus non-PCs CD45 positive events as the
denominator (8). Reporting PC levels as a percentage of
nucleated cells is likely to offer a better comparison with
molecular techniques than reporting as a percentage of
leucocytes because DNA is typically isolated from all
nucleated cells. However, different lysing approaches (e.g.
ammonium chloride vs. fixative-based lysing approaches)
can affect the number of total nucleated cells because
ammonium chloride will also lyse some nucleated ery-
throid cells, while using total leucocytes as the denomina-
tor is not affected by different lysing agents. The impact
of different reporting or lysing approaches is likely to
have a negligible effect on the level of MRD detected but
the report must clearly state which method is used.
Although multicentre data has shown that while using
0.01%/1024 as the limit of detection (LOD) for MRD
monitoring by flow is of clear prognostic value, it does
Cytometry Part B: Clinical Cytometry
 GUIDELINES ON MYELOMA MRD ANALYSIS AND REPORTING
Table 1
Estimated LOD and LLOQ According to the Total Number of
Events Acquired
Total number of
cells analyzed LOD (%) LLOQ (%)
100,000 0.03 0.05
200,000 0.015 0.025
500,000 0.006 0.01
1,000,000 0.003 0.005
2,000,000 0.0015 0.0025
3,000,000 0.001 0.0017
5,000,000 0.0006 0.001
LOD 5 (30/Total cells) 3100%; LOQ 5 (50/Total cells) 3100%.
not yet translate into a plateau (8,19,21). In MM as in
other diseases, it is almost certain that as treatment regi-
mens continue to improve, the threshold of 0.01%/1024
is likely to become less relevant. The consensus is that
current and future MRD analyses should target a lower
LOD preferable of < 0.001%/1025 (18,22), which
requires acquisition of at least 3 3 106 bone marrow
cells; ideally a limit of quantification of 0.001% should
be targeted in the new MM flow-MRD approaches which
requires that a minimum of 5 3 106 BM cells are
acquired. If it is required that a categorical assessment
(MRD-positive or MRD-negative) is provided, it is impor-
tant to state the threshold used and ideally reference the
threshold appropriately, e.g. MRD-negative at the 0.01%
level [EMN guidelines (2)].
Reporting MRD levels and the limit of detection (LOD) and
the lower limit of quantification (LLOQ)
As discussed above, the use of statements such as MRD-
positive or MRD-negative can be poorly informative if
there is not a universally agreed threshold defined for
(uniform) response criteria. Although many laboratories
are currently performing more sensitive flow-MRD assays
(i. e.:0.001%/1025), a threshold of 0.01%/1024 is currently
accepted as being the minimum clinically relevant and
routinely achievable threshold. In order to ensure that
MRD results are informative, even if the accepted thresh-
old for MRD detection will likely change in the future
(e.g. down to 0.001%/1025), it is critical that the MRD
report includes sufficient information to determine the
detection and quantification limits of the individual assay,
used for any individual sample. Such limits are based on
four major parameters: (i) the total number of events ana-
lyzed, (ii) the number of neoplastic PC events detected
within them, (iii) the smallest number of events required
to reproducibly detect a population of neoplastic PCs
and, (iv) the smallest number of events required to repro-
ducibly quantify a population of neoplastic PCs.
The College of American Pathologists requires that each
laboratory confirm the lower LOD and LLOQ for each test
(23–25). There are numerous studies demonstrating that
20 events is a conservative value for the smallest (homoge-
neous) population that can be detected in a given flow
cytometric list mode data file by experienced operators
(26,27). Therefore, the maximum number of events that
Cytometry Part B: Clinical Cytometry
37
could be considered to be below the limit of detection is
19 events, but due to the potential counting errors (95%
confidence interval for a count of 19 events would be of
11 to 30 events) this implies that the LOD can be estimated
as (30/total number of cells analyzed) 3 100%. Similarly, it
is also widely accepted that more than 50 events is a stand-
ard threshold for reproducible enumeration of a cell popu-
lation by experienced flow cytometer operators (28,29);
consequently, the limit of quantification can be estimated
as (50/total number of cells analyzed) 3 100%. Thus, the
LOD and the LLOQ will be both typically dependent on
the total number of cells analyzed; the expected limit of
detection and lower limit of quantification for a range of
cells analyzed are shown in Table 1.
Reporting both the number of neoplastic PCs identi-
fied in a sample and the total number of (normal 1 neo-
plastic) events contained in it, is mandatory when
expressing the level of sensitivity of the flow-MRD assay
performed in an individual sample. Thus, a complete
report would include the percentage of neoplastic PCs
from the whole sample cellularity, either the LOD or
LLOQ or both, depending on the MRD levels, and ideally
also the total number of cells evaluated for that sample,
after excluding doublets and debris. For example, if neo-
plastic PCs are not detected, then the minimal data
required includes the LOD of the assay, including ideally
also the total number of cells evaluated, e.g.: neoplastic
PCs < 0.0004% (neoplastic PCs < 20 events; total cells
analyzed 5 5,000,000). In turn, if neoplastic PCs are
detected above the LLOQ, then the minimal data
required is the percentage of neoplastic PCs and the
LLOQ, ideally including also the number of events (neo-
plastic PCs and total cells) measured, e.g.: neoplastic
PCs 5 0.05% of the cells (LLOQ 0.0025%; neoplastic
PCs 5 1,000 events; total cells analyzed 5 2,000,000).
Finally if neoplastic PCs are detected at a level between
that of the LOD and the LLOQ, then a percentage MRD
cannot be reported; instead the lower and upper limits
are reported, e.g.: neoplastic PCs detected below the
quantitative range (0.0004 2 0.001%; neoplastic
PCs 5 35 events, total cells analyzed 5 5,000,000). The
proportion of neoplastic PCs within the total plasma
cell compartment should always be reported for two
reasons: first, the information might be of prognostic
relevance (30); secondly, because the balance between
phenotypically aberrant and normal PCs is not likely to
be affected significantly by hemodilution, therefore pro-
viding a measure about the quality of the sample.
SUMMARY LIST OF POTENTIAL PITFALLS OCCURRING
DURING MRD DATA ANALYSIS
 Gating errors associated with inclusion of non-plasma
cell events or exclusion of neoplastic PCs (please see
PC gating above).
 Variation (e.g.: increase) in minor antigen profiles on
normal PC subsets (e.g.: CD192, CD452, and
CD561); please note that there are no specific
38 ARROZ ET AL.
individual tumor antigens, and one marker alone is
typically not enough to define neoplastic PCs.
 Presence of minor PC subpopulations that only
become apparent with the acquisition of >106 BM
cells.
 Phenotypic heterogeneity of neoplastic PCs at diagno-
sis should be taken into account to differentiate anti-
gen shifts within neoplastic PCs (e.g.: loss of CD56),
from phenotypic selection of chemo-resistant subclones
(antigen restriction rather than shift).
 Neoplastic PCs do not have predictable FSC/SSC and
CD45/SSC profiles and they often show substantial
autofluorescence.
SUMMARY OF MINIMUM RECOMMENDATIONS
 Minimum requirements for flow-MRD monitoring in
MM include the usage of at least six-color antibody
combinations in a single tube, most preferably eight, a
gating strategy based on simultaneous assessment of
CD38, CD138, and CD45 expression to maximize PC
inclusion whilst minimizing contamination by other
cells along with the use of FSC and SSC to exclude
cell doublets and debris, and at least one combination
that simultaneously includes CD38/CD45/CD19/
CD56.
 Interpretation of antigen expression profiles should
be performed with caution as many of the reported
aberrancies based on 1-2D marker analysis are also
present in specific (e.g., minor) subsets of normal
PCs.
 The capacity to perform additional analysis of samples
that require further evaluations by either acquisition
of more events and/or characterization of further anti-
gens namely CyIgj/CyIgk on the same sample while it
still remains relatively fresh, is recommended in cen-
ters that perform MRD assessment.
 The role of cytoplasmic immunoglobulin light chain
j/k expression for high-sensitive MRD detection
(1025) is currently being evaluated.
 The report should include the MRD level plus the
LOD and LLOQ of the assay, the proportion and num-
ber of neoplastic and normal PCs (if present) within
the BM PC compartment, the immunophenotypic
expression profile of neoplastic PCs, as well as cell
viability and the quality of sample (e.g.: percentages
of B-cell precursors, nucleated red cells, and/or mast
cells to reflect potential hemodilution based on the
absence of such BM-specific cell populations).
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